Electrophoresis is not a new art, the first process of which was a moving boundary type described by Tiselius in 1937. It was used to study the movement and interactions of proteins. Since then, many methods have been devised for carrying out the process, and many papers have been published describing these processes.
To date, continuous flow electrophoresis devices have typically used a rectangular chamber having a very thin cross section which ranges from 0.05 to 0.15 cm. An electrolytic buffer solution is injected at one end of the chamber and drawn off at the opposite end, simultaneously filling and creating a flow through the chamber. Positive and negative electrodes are positioned opposite each other along the thin sides of the chamber, causing an electrical field to be impressed laterally across the width of the chamber. A sample stream containing a homogenous mixture of variously sized organic particles having different electrokinetic properties is injected at the same end of the chamber as the buffer so that it flows with the buffer through the chamber. As the sample stream flows through the chamber, the charged particles migrate toward oppositely charged electrodes a distance which is determined by the size of a particle, the viscosity of the buffer, and the strength of the charge on a particle. This causes species of particles to form bands across the width of the chamber, which are collected by a collection array along with a quantity of buffer commensurate with the sum of the injected buffer and sample. Thus, by continuously injecting a buffer and sample stream of particles, a continuous collection of fractionated species particles (and buffer) is accomplished.
Problems arise, however, because flow dynamics in the aforementioned chamber cause distortions of the separated species particles. One of these distortions, known as Poiseuille flow, is induced from frictional effects of the walls of the chamber on adjacent fluid flow. This causes particles flowing along the chamber walls to move slower and reside in the chamber for a longer period of time than particles flowing along the mid plane of the chamber, allowing the slower particles to migrate laterally a further distance than mid plane particles. Thus, when viewed from the collection end of the chamber, a crescent-shaped band is formed, with the nose of the crescent generally in the mid plane of the chamber and the tails of the crescent nearer their respective walls.
A second flow distortion, known as electroosmosis, is a bidirectional lateral flow across the width of the chamber. This is caused by a phenomenon known as the zeta potential, which generally is due to a weak negative charge at the chamber walls when a hydrated ionic solution is present. The negatively charged walls in turn attract positive ions from the solution, which form a layer of positive ions on the surfaces of each of the chamber walls. Being only weakly attracted to the walls, the positive ions are pulled along the chamber walls under the influence of the negative electrode, which generates a fluid flow along the walls in a direction toward the negative electrode. Because the sides of the chamber are closed, a corresponding counterflow in the mid plane of the chamber, in an opposite direction, is generated which causes the injected sample stream to form a parabolic crescent, with the nose of the crescent (generally in the mid plane of the chamber) to be pointed toward the positive electrode, while the tails of the crescent (along the walls) are drawn toward the negative electrode. A more thorough discussion of these problems may be found in Strickler, A. and Sacks, T. Preparative Biochemistry, 3, p. 269-277 (1973).
These two aforementioned problems, Poiseuille flow and electroosmosis, combine in a conventional continuous flow electrophoresis device to form crescent-shaped distortions of the separated species particles which makes the collection of the separated particles difficult, if not impossible. Several attempts to compensate or eliminate these effects have been tried but have proved to be ineffective or impractical. While these methods can bring one species band of particles into focus, to date, none have been demonstrated which are able to bring all species bands into focus simultaneously. One such method to eliminate flow disturbances was proposed by A. Kolin and B. L. Ellerbroek in a paper entitled "Theory of Simultaneous Multiple Streak Collimation in Continuous-Flow Electrophoresis by Superposition of Electro-Osmosis and Thermal Convection," Separation and Purification Methods, 8, 1-19 (1979). This method utilizes a cross flow to neutralize electroosmosis and uses thermal convection to dampen the Poiseuille effect so that the center plane region of the chamber will be relatively distortion-free. The problem with this scheme, however, is that exact calibration of the counter flow to compensate for electroosmosis throughout the length of the chamber is virtually impossible to achieve.
Another proposed method for compensating for flow disturbances in continuous flow electrophoresis devices is set forth in Strickler, A. and Sacks, T., "Focusing in Continuous Flow Electrophoresis System by Electrical Control of Effective Cell Wall Zeta Potential," Annals of New York Academy of Science, 209 (1973). This scheme consisted of coating longitudinal sections of the chamber with various coatings having different zeta potentials and having separate electrodes for each differently coated section. The electrical field in each section may be independently varied as a sample passes through the chamber, thus compensating for flow distortions in fractionated species acquired in a prior coated section. Although this method is workable, it is necessary to control the process by visual observation, which is operator intensive and therefore somewhat impractical.
Yet another electrophoresis device is disclosed in U.S. Pat. No. 4,358,358 which uses a pair of endless, wide Mylar.TM. belts which form the broad walls of an electrophoresis chamber which is supported in a tank of buffer solution. The surfaces of these belts which face the chamber are coated with Methylcellulose which has a zeta potential near zero. The belts are synchronized to simultaneously move across the electrophoresis chamber at the same rate as the buffer flow, which entrains the buffer to flow through the chamber as a rigid medium, eliminating problems associated with Poiseuille flow. In theory, this is a workable system because there would be no flow distortions to compensate for and no electroosmosis flow because there would be no charge on the moving walls because of the Methylcellulose coating. It has been discovered, however, that Methylcellulose and other wall coatings are not particularly stable, resulting in difficulty in maintaining a zero zeta potential coating. Additionally, any contamination of the coating by the sample increases the zeta potential of the coating. Further, contact between the wall coating and the necessary seals and reels tends to have an abrasive effect on the wall coatings, causing an increase in the zeta potential. Still further, the chamber and belt drives were immersed in an enclosure containing an electrolytic solution, which presented operational inconveniences. As a result of these problems, the device disclosed in U.S. Pat. No. 4,358,358 is difficult to operate reliably.
Accordingly, it is an object of this invention to provide a moving wall, continuous flow electrophoresis device which will, by entraining the buffer to flow as a rigid body through the electrophoresis chamber, eliminate flow distortions from the Poiseuille effect.
Another object of this invention is to provide a moving wall electrophoresis device which will exactly compensate for electroosmosis flow by a simple mechanical adjustment, bringing all fractionated species particles into focus simultaneously.
Yet another object of this invention is to provide a moving wall, continuous flow electrophoresis device which is not operator intensive.